DESCRIPTION (Verbatim from the application): The transmembrane ligand ephrinB2 is specifically expressed by arterial but not venous endothelial cells in both embryos and adults, whereas its receptor EphB4 is conversely expressed by veins but not arteries. Both the ligand and receptor are essential for embryonic cardiovascular development. The objective of this proposal is to understand more precisely the function of ephrinB2-EphB4 signaling in angiogenesis. Using conditional loss-and gain-of-function manipulations in knockout and transgenic mice, we will test the hypothesis that bi-directional signaling between arteries and veins mediated by this ligand-receptor pair is essential for angiogenesis, and ask whether the cellular function of this signaling is primarily attractive or repulsive. In Specific Aim I, we will knock out ephrinB2 specifically within endothelial and endocardial cells, to determine whether its essential function is indeed exerted within the circulatory system. These studies will be complemented by experiments to selectively rescue the ephrinB2 knockout phenotype within the circulatory system. Temporally controlled, pan-endothelial knockout of ephrinB2 will also be performed, to determine whether the gene is also required at later stages of angiogenesis. In Specific Aim II, we will use both in vitro embryoid body assays of blood vessel formation and in vivo embryo chimeras in conjunction with heterozygous and homozygous ES cells mutant for ephrinB2 or EphB4 to distinguish whether the primary requirement for these genes in early cardiovascular development is in the heart, the peripheral vasculature, or both. In Specific Aim III we will perform constitutive and/or conditional pan-endothelial expression of ephrinB2 and EphB4 transgenes to determine whether the arterial- and venous-specific expression, respectively, of these genes is essential for their proper function in the circulatory system. In Specific Aim IV we will create lines of "dual-indicator" mice that can be used to simultaneously distinguish arteries and veins using genetically encoded photochemical markers. These mice will be used in both in vivo and in vitro experiments to further study the role of cell-cell interactions between arteries and veins and the role of ephrinB2-EphB4 signaling in mediating these interactions. Mechanistic studies of the role of ephrinB2-EphB4 signaling in development are highly like to inform our understanding of its function in adult angiogenesis, and may suggest new therapeutic strategies for the inhibition or promotion of angiogenesis in clinical settings such as cancer and heart disease, via pharmacological manipulation of these artery- and vein-specific signaling molecules.